T cells or T lymphocytes belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells, by the presence of a special receptor on their cell surface called T cell receptors (TCR). Several different subsets of T cells have been discovered, each with a distinct function including T helper cell (TH cells), Cytotoxic T cells (TC cells, or CTLs), Memory T cells (TM cells) including central memory T cells (TCM cells) and effector memory T cells (TEM cells), Natural killer T cells (NKT cells), gamma delta T cells (γδT cells), and regulatory T cells (Treg cells).
Regulatory T cells, also known as suppressor T cells, are a specialized subpopulation of T cells that act to suppress immune responses of other cells. For example, Treg cells play a major role in suppressing T cell-mediated immunity during an immune reaction and in suppressing auto-reactive T cells that escaped the process of negative selection in the thymus. As such, Treg cells provide an important “self-check” to prevent excessive immunogenic reactions and are thus crucial for the maintenance of immune system homeostasis and tolerance to self-antigens.
Two major classes of Treg cells are the naturally-occurring Treg cells and the adaptive Treg cells. Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Adaptive Treg cells (including Tr1 cells, Th2 cells, Th3 cells, and Th17 cells) appear to originate in the periphery during a normal immune response and seem to promote Treg cell development and function.
Adaptive Treg cells share many of the attributes of naturally-occurring Treg cells but can differ in critical cell surface biomarkers and functional attributes. For instance, Tr1 and Th3 cells have been described that produce IL-10 and TGFβ, respectively. These results have led to novel approaches to immunotherapy as the ability to isolate, enrich, and expand this cell subset in mice has led to novel therapeutic interventions in immunological diseases.
The identification of naturally-occurring Treg cells as an important component of self-tolerance has opened a major area of investigation in immunology and the basic process that control immune tolerance. Regulatory T cells have a unique and robust therapeutic profile. The cells require specific T cell receptor (TCR)-mediated activation to develop regulatory activity but their effector function appears to be non-specific, regulating local inflammatory responses through a combination of cell-cell contact and suppressive cytokine production. Numerous studies have demonstrated the potent influence of naturally-occurring Treg cell in suppressing pathologic immune responses in autoimmune diseases, transplantation, and graft-vs-host diseases. However, a major obstacle to the study and application of naturally-occurring Treg cells in the human setting has been the lack of specific cell surface biomarkers to define and separate Treg cells from other subsets of T cells such as, e.g., TH cells, TC cells, TCM cells, TEM cells as well as to distinguish between different subpopulations of Treg cells.
A number of different methods are employed in research to identify, isolate, enrich, or otherwise exploit Treg cells. The most widely used markers for naturally-occurring Treg cells are cluster of differentiation 4 (CD4), cluster of differentiation 25 (CD25), forkhead/winged-helix transcription factor box P3 (FoxP3), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR), lymphocyte activation gene-3 (LAG-3), and cluster of differentiation 127 (CD127). Unfortunately, accumulating evidence suggests that the above-listed markers are not strictly Treg cell-specific. For example, high expression of CD25 and CD4 surface markers (CD4+CD25+ cells) was originally used to identify naturally-occurring Treg cells. However, CD4 is also expressed on TH cells, and a subpopulation of TM cells. CD25 is also expressed on non-regulatory T cells in the setting of immune activation such as during an immune response to a pathogen. Thus, as defined by CD4 and CD25 expression, Treg cells comprise about 5-10% of mature TH cells. The additional measurement of cellular expression of Foxp3 allowed a more specific analysis of naturally-occurring Treg cells (CD4+CD25+FoxP3+ cells). However, Foxp3 is also transiently expressed in activated TEM cells and it is now well documented that most human CD4+ and CD8+ T cells transiently express Foxp3 upon activation, including CD4+ CD25low/− T cells, TH cells, TC cells, and memory T cells. Furthermore, FoxP3 is a nuclear marker requires cell membrane permeabilization prior to staining. As such, use of this biomarker precludes subsequent processing steps such as separating, isolating, enriching, or expanding viable naturally-occurring Treg cells for functional studies or for use in an immunotherapy.
It has been suggested that the addition of CD127 could be used to discriminate between naturally-occurring Treg cells (which exhibit a CD127low/− expression pattern) and TH cells (which exhibit a CD127+ expression pattern) in humans. However, it has been recently reported that most CD4+ T cells down-regulate CD127 upon activation. Furthermore, loss of CD127 is a characteristic feature of T follicular helper cells (TFH cells), which provide help for B cells in human tonsils. CTLA-4 is a negative regulator of T-cell activation, which is up-regulated on all CD4+ and CD8+ T cells, 2-3 days following activation. Similarly, the expression of GITR and LAG-3 is induced in TEM cells upon activation. Thus, all the presently-used Treg cell biomarkers (CD25, CTLA-4, GITR, CD127, LAG-3, GARP and FoxP3) appear to be general T-cell activation markers. As such, these biomarkers do not appear to be Treg-cell-specific and therefore are not reliable for distinguishing naturally-occurring Treg cells from other T cell subsets like activated TH cells. Thus, it is likely that many of the natural and adaptive regulatory T cells are missed in current biomarker studies, calling into question the conclusions related to deficiencies or defects in certain autoimmune settings.
Thus there is a need to develop improved methods of identifying, isolating, enriching, and expanding a population of immunosuppressive regulatory T-cells as well as methods of modulating an immune reaction in an individual. Additionally, there is a need to develop compositions comprising a population of immunosuppressive regulatory T-cell as well as uses of such compounds for modulating an immune reaction in an individual. Lastly, there is a need to develop kits comprising biomarkers and/or other components useful to conduct the above described methods.